A mutant D-amino acid aminotransferase with broad substrate specificity: construction by replacement of the interdomain loop Pro119- Arg120-Pro121 by Gly-Gly-Gly |
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Authors: | Gutierrez A; Yoshimura T; Fuchikami Y; Soda K; Esaki N |
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Affiliation: | Institute for Chemical Research, Kyoto University, Uji, Japan. |
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Abstract: | D-amino acid aminotransferase (EC 2.6.1.21) catalyzes the interconversion
of various D-amino acids and 2-oxo acids. Each homodimer subunit consists
of two domains, which are connected by a single loop,
Asn118-Pro119-Arg120-Pro121. The loop has no direct contact with the active
site region or the cofactor, pyridoxal 5'- phosphate. We attempted to
increase the conformational flexibility of this loop through a triple
glycine substitution. The resultant mutant P119G-R120G-P121G has features
clearly different from the wild-type enzyme under overall as well as
half-reaction conditions. The pre- steady-state kinetic analyses of half
reactions showed that the mutant enzyme has kmax values higher than the
wild-type enzyme towards most D- amino acids examined. A concomitant
decrease in substrate affinity (1/Kd), particularly for acidic amino acids,
was also observed. A putative binding site for the distal carboxyl group of
acidic amino acids in the wild-type enzyme was incidentally displaced by
the loop mutation, indicating a functional linkage between the interdomain
loop and the active site region. This study has exemplified the usefulness
of engineering relatively distant loops as a means to modify substrate
specificity of an enzyme.
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